Bioprinted hASC‐laden collagen/HA constructs with meringue‐like macro/micropores

Abstract Extrusion‐based bioprinting is one of the most effective methods for fabricating cell‐laden mesh structures. However, insufficient cellular activities within the printed cylindrical cell‐matrix blocks, inducing low cell‐to‐cell interactions due to the disturbance of the matrix hydrogel, remain to be addressed. Hence, various sacrificial materials or void‐forming methods have been used; however, most of them cannot solve the problem completely or require complicated fabricating procedures. Herein, we suggest a bioprinted cell‐laden collagen/hydroxyapatite (HA) construct comprising meringue‐like porous cell‐laden structures to enhance osteogenic activity. A porous bioink is generated using a culinary process, i.e., the whipping method, and the whipping conditions, such as the material concentration, time, and speed, are selected appropriately. The constructs fabricated using the meringue‐like bioink with MG63 cells and human adipose stem cells exhibit outstanding metabolic and osteogenic activities owing to the synergistic effects of the efficient cell‐to‐cell interactions and HA stimulation released from the porous structure. The in vitro cellular responses indicate that the meringue‐like collagen bioink for achieving an extremely porous cell‐laden construct can be a highly promising cell‐laden material for various tissue regeneration applications.


| INTRODUCTION
Bioprinting for fabricating cell-laden porous scaffolds has become one of the most important processes in tissue engineering. [1][2][3][4][5] As reported by several researchers, cell-laden scaffolds offer numerous advantages, such as versatility in efficiently loading different cell types in a desired position and ease in cell density control compared with conventional scaffolds seeded with cells. [1][2][3][4][5] Although bioprinted cellladen scaffolds demonstrate outstanding biological activities, they present shortcomings that must be solved. For example, cells encapsulated in a bioink exhibit limited cellular responses because of the low porosity within printed cell-laden struts, thereby inducing low cell-to-cell or cell-to-biomaterial interactions; additionally, the cellladen structure cannot sustain its complex three-dimensional (3D) geometry owing to the inferior mechanical properties of the matrix material, i.e., hydrogel. [6][7][8] Recently, numerous studies have been performed to overcome the limitation of the cell construct and fabricate a cell-laden scaffold with hierarchical (macro and micro) porous structures; the typical method involves the use of various sacrificial materials. [7][8][9][10][11][12] For instance, pluronic block copolymers (PF-127) were used as a leaching material in alginate-based bioink laden with human mesenchymal stem cells (hMSCs). 12 The degradation of PF-127 micelles successfully formed a microporous structure in the crosslinked hMSC-laden alginate scaffold. However, the hybrid bioink is thermodynamically unstable because of the properties of PF-127; in fact, PF-127 has been reported to be cytotoxic under certain conditions, including a critical micelle concentration. 13 Bao et al. attempted to overcome the low porosity of a cell-laden construct by printing chitosan/poly(ethylene glycol) mixture in a phase-separation inducing matrix (PSIM) to trigger the micropore formation. 8 The printed structure exhibited a homogeneous and hierarchical interconnected porous structure and demonstrated relatively high cell viability, proliferation, and migration compared with conventional cell-laden structures. However, the multistep fabrication procedure (i.e., PSIM preparation, emulsion printing, and triggering process) to achieve the porous cell-laden construct was extremely complicated. In our previous study, we described a biofabrication method for solving the limited porosity of cell-laden constructs by controlling the diameter of printed struts, which resulted in a 3D mesh structure. 14 As expected, the constructs with thinner cellladen struts showed significantly higher cell-metabolic activities than those with thicker cell-laden struts. However, the diameter of the cellladen strut was limited to $100 μm owing to the limitations of extrusion-based printing using a nozzle. 8 In addition, the reduced diameter was only effective in one dimension (the radial direction from the inside of the strut); hence, the cellular enhancement of the encapsulated cells was limited. The detailed summarized methods to fabricate cell-laden structures with microscale pore geometries are described in Table S1 in Appendix S1.
Considering the limitations of conventional bioprinting methods for achieving the cell-laden porous construct, we focused on the scientific principle of a meringue, i.e., a firm and sustainable foam structure containing nanofibrillated egg white albumin created via whipping. 15 In the culinary field, food scientists have been investigating the principle of foaming. [15][16][17][18][19] Whipping method involves stirring using a whipper to develop a foam structure by mixing and simultaneously embedding air into a liquid. 20,21 Generally, the foam structure is unstable owing to the high surface tension at the interface of air bubbles and liquid. 15,16,22 In other words, lowering the surface tension of interfaces using surfactants or proteins can stabilize the foam structure. 16,17,23 Generally, proteins, which are composed of various types and combinations of amino acids, exhibit hydrophilic and hydrophobic characteristics (similar to surfactants). As such, a viscoelastic fibrous wall can be formed and the steady capture of air bubbles facilitated. [15][16][17][24][25][26] Consequently, air bubbles can be entrapped in the structure. Therefore, meringue has been referred to as a "culinary scaffold" by food scientists owing to its firm aerated texture. [15][16][17][18][19][27][28][29][30][31][32][33] In this study, we prepared a meringue-like porous structure, which can be fabricated easily via whipping, to be used as a tissue regenerative cell-laden construct. Accordingly, we used a collagen solution to fabricate a porous cell-laden structure. Based on the effects of the material and processing parameters, such as collagen concentration and stirring time and speed, on the pore size and porosity, the optimal processing conditions were selected to fabricate a meringue-like collagen structure (hereinafter, CM is used to represent collagen-meringue) with sufficiently viable cells. To verify the advantages of the CM structure, human osteoblast-like cells (MG63) and human adipose-derived stem cells (hASCs) were used in the collagenbased structures, and the cell viability, cell migration, and osteogenic activities were examined in vitro. Moreover, hydroxyapatite (HA) was incorporated into the CM structure to further induce osteogenic activities. This physically and biologically enhanced collagen-based meringue-like structure can be a highly potential biomaterial for various tissue regenerative applications.

| Preparation of cell-laden collagen constructs
A neutralized collagen solution (5 w/v%) was mixed with hydroxyapatite (HA; 10 w/v%) and cells (1 Â 10 6 cells ml À1 ) and then printed using a conventional temperature-controlled printing system. 34 The details of materials and the preparation process of the conventional cell-laden constructs are described in Appendix S1. Meanwhile, collagen solutions with various concentrations (2, 3, 4, 5, and 6 w/v%) were mixed with the 1-mM genipin solution at a 7:3 volume ratio (final volume: 1 ml) and stirred using an automatic stirrer equipped with a 3D whipper that was designed to fit in a 50-ml conical tube [ Figure 1f]. The stirring was performed at 500, 1000, 1500, 2000, 2500, and 3000 rpm for 5, 10, 15, 20, 25, and 30 min at 25 C to examine the optimal whipping condition. Subsequently, the whipped collagen was gently mixed with the cells (1 Â 10 6 cells ml À1 ) using a three-way stopcock. The cell-laden collagen meringue-like bioink was injected into polydimethylsiloxane cylindrical molds and incubated in the culture media containing genipin (1 mM) for 1 h at 37 C for additional crosslinking. Non-porous cell-laden collagen bioink without whipping (NC) was injected and incubated using the same approach to perform a comparison with the meringue-like structure (CM). Collagen/HA composite scaffolds were prepared by mixing HA powder (10 w/v%) with collagen solutions and were subsequently either printed (CHP) or whipped (CHM). The abbreviation and the actual compositions of the bioinks and structures are explained in Table S2 in Appendix S1 as well.

| Characterization of collagen meringue-like bioinks
The porous structure of the whipped collagen was captured using a digital camera connected to a microscope (BX FM-32; Olympus).
Using the optical images obtained, the number of bubbles and the diameter of bubbles were measured via the ImageJ software (National Institutes of Health). The air volume fraction was determined by measuring the total volume of the whipped collagen using the rotating speed and time and then comparing it with the initial volume of the collagen solution (5 ml). Characterization of HA-supplemented F I G U R E 1 Legend on next page. collagen meringue bioinks (CHM) was performed using EDS-SEM, XRD, and TGA as described in Appendix S1.
The rheological properties of the normal collagen (NC and dNC) and collagen meringue-like (CM and CHM) bioinks were assessed using a rotational rheometer (Bohlin Gemini HR Nano; Malvern Instruments) with a cone-and-plate geometry (40- Table S3 in Appendix S1. The detailed RNA extraction and transcription process is also described in Appendix S1.

| In vitro cellular analysis
Osteopontin (OPN) antibody immunofluorescence was analyzed to evaluate osteogenesis after 21 d of culture. Briefly, the cell-laden constructs were treated with anti-OPN primary antibody (1:200 in PBS; Invitrogen) overnight at 4 C after fixation. Subsequently, the samples were incubated with DAPI and secondary anti-rabbit antibody (1:500 in PBS; Invitrogen) conjugated with Alexa Fluor 488 for 1 h. Fluorescence images were obtained using a confocal microscope and evaluated by the ImageJ software.

| Statistical analysis
Data are presented as mean ± SD. All statistical analyses were performed using Instat 3 (GraphPad Software), and differences were considered statistically significant when p < 0.05. Independent t-tests were performed for the results of the two groups. For pairwise comparisons of results involving more than two groups, the one-way analysis of variance was performed, followed by Tukey's multiple comparisons test.

| RESULTS AND DISCUSSION
In the study, we adopted the principle of a meringue, a firm and sustainable sponge-like structure composed of egg whites that is prepared via whipping, to achieve a highly porous cell-laden structure. Figure 1a shows the meringue structure composed of hydrophilic and hydrophobic regions in the interfacing fibrous wall; it implies that macro/microsized air bubbles in a protein solution can be entrapped stably. 16,17,[24][25][26] In this study, collagen was used to fabricate the meringue-like foam structure because the collagen fibrous protein molecules are composed of hydrophobic (glycine and proline) and hydrophilic amino acid (hydroxyproline) [ Figure 1b]. 35,36 Hence, the protein molecules of collagen are expected to be entrapped between the air bubbles and aqueous solution, forming an interfacial fibrous wall through the whipping process [ Figure 1c-e], similar to the mechanism of egg white protein formation in the culinary field. 15 To achieve a meringue-like structure, the whipper shown in Figure 1f was rotated using an automatic stirrer to control the rotating speed such that the whipping process can be analyzed quantitatively.
In this study, a solution (type-I collagen 4 w/v% and 1-mM genipin) was used to form a meringue-like structure with various pore geometries via whipping (2000 rpm for 20 min). In the collagen solution, 1-mM genipin was mixed at a volume ratio of 3:7 (genipin:collagen) for the preliminary crosslinking of the collagen solution to enhance the sustainability of the pore structure [ Figure 1g]. Finally, the sponge-like collagen solution was gently mixed with the cells (density: 1 Â 10 6 cells ml À1 ) using a three-way stopcock [ Figure 1h]. Compared

| Rheological properties of collagen meringue
Generally, bioinks, which can be used in 3D bioprinting or any injectable processes to repair damaged tissues, exhibit rheological properties that can maintain complex structures after processing is completed. 40 In this study, we prepared a 4 w/v% concentration colla-  Figure S1. This shows that the CM bioink possessed much better self-recovery than the typical collagen bioink.

| Cell activation of MG63-laden collagen meringue
To observe the cellular activities of the CM structure, MG63 cells in Appendix S1 (also see Figure S3), indicating that the highly porous CM structure induced cell proliferation and osteogenic differentiation, unlike the NC structure, owing to the efficient transport of nutrients, oxygen, and metabolic wastes by the macro/microporous structure.

| HA-assisted CM structure
In general, collagen and HA composites have been extensively investigated in bone tissue engineering because they are the main constituents of human bone tissues. [44][45][46] In particular, collagen has been used as a scaffold for regenerating bone tissues owing to favorable biochemical properties. 47 However, the unfavorable mechanical (h) Cell number per unit area (1.25 Â 1.25 mm) and (i) F-Actin area/mm 2 measured using DAPI/phalloidin images, after 14 d of cell culture. NS: Statistical nonsignificance; *p < 0.05, **p < 0.005, and ***p < 0.0005 properties of the collagen prevent its use as a hard-tissue engineering material. Hence, various collagen composite systems supplemented with osteoconductive bioceramics, such as HA and tricalcium phosphates, have been applied to enhance the mechanical properties and promote biological activities. 48 In this study, bioceramic HA was incorporated into the collagen solution to develop the composite structure, i.e., the CHM structure ( Figure 5). As a result, HA composition of 10 w/v% in the collagen meringue bioink exhibited stable foam structure and favorable cell viability. The detail is described in Appendix S1.

| Comparisons of in vitro cellular activities for conventionally bioprinted and meringue-like structures
To demonstrate the feasibility of the meringue-like structure, we used a conventionally bioprinted cell-laden collagen (5 w/v%)/HA (10 w/v%) mesh structure with uniform macropores for the comparison of the conventional mesh porous structure and the whipped meringue-like porous structure. A collagen concentration of 5 w/v% has been widely used in the conventional bioprinting process because the rheological properties afforded by this concentration are appropriate for fabricating cell-laden structures in terms of printability and in situ cell viability after bioprinting. 34,41 In the cell-laden structures, we incorporated hASCs, which have been extensively used to evaluate osteogenic activities in various scaffolds. [49][50][51][52] The cytocompatibility of the CM structure for hASCs was confirmed as described in Appendix S1 (also see Figure S4).  Table S4. Figure 6b shows a process diagram illustrating the stable sustainability of the porous meringue structure after printing and reasonable cell viability ($90%) for the typical printing parameters, pneumatic pressure, and nozzle diameter. The results show the following three typical regions: "Ο," stable air bubble sustainability after printing and reasonable cell viability; "Δ," unstable flow due to the relatively low pneumatic pressure; and "Â," bubble destruction inducing relative larger average pore diameters (over 270 μm) and low cell viability. Using the process diagram, the appropriate printing parameters can be selected to achieve a stable porous meringue-like cell-laden structure.
The pores also seemed interconnected, as indicated in red arrows and dotted lines on the SEM images. Figure 6e shows the live/dead images (on day 3) of the CHP and CHM structures, and the cell viability for both structures was approximately 90%, indicating that the bioprinting process was completely safe for the laden cells [ Figure 6f]. The results indicate that the CHM structure can afford a much more favorable cellular environment to promote highly active cell-cell interactions than the CHP structure. This is attributable to the homogenously distributed porous structure, which was achieved using the meringue process. The results of in vitro cellular activities show that the interconnected macro/microporous structure (i.e., meringue-like structure) can provide a highly efficient microcellular environment, such as efficient cell-cell interactions and more active HA stimulation to the laden cells, to enhance the osteogenic activities of the embedded hASCs, as compared with the conventional bioprinted structure; this implies that the meringue-like cell-laden structure may be a highly potential bioactive platform for the regeneration of bone tissues.

| Application of cell-laden CM to various porous molds
As mentioned previously, the utility of collagen in hard-tissue engineering can be challenging owing to the unfavorable mechanical properties of collagen; therefore, the collagen structure was supplemented with other synthetic biomaterials to enhance its mechanical properties. 56 Although the rheological properties were enhanced by the whipping process and the composite system with HA, the meringue structure still required mechanical reinforcement for further preclinical application to hard-tissue regeneration, such as implantation into a 3D complex bone defect. Therefore, we developed various types of mechanically reinforcing porous molds to support the injectable CM or its composite bioink in a complex 3D construct ( Figure 8) as described in Appendix S1 (also see Figure S5) in detail. Based on the results, we believe that the combinational cell-laden structure using the porous molds can be a potential structure to be applied to various load-bearing regions of hard-tissue regeneration.

| CONCLUSION
In this study, we developed a biofabrication process for 3D cell-laden porous collagen/HA scaffolds with well-interconnected macro/micropores. To achieve a meringue-like porous structure, various material/ processing parameters, such as collagen concentration, whipping speed, and time, were selected appropriately. This whipping method permits not only the homogeneous encapsulation of osteoblast-like cells or hASCs but also mechanically stable pore structures. This enables high cell viability and proliferation within several millimeters of the structure owing to the efficient transport of nutrients and oxygen. The assessment of in vitro cellular activities using hASCs verified that high expression levels of osteogenic genes, such as COL1, BMP2, OCN, ERK1/2, and p38 MAPK, were observed for the meringue-like collagen/HA structure compared with the conventionally bioprinted collagen/HA mesh structure because of the synergistic effects of the efficient cell-cell interactions and HA stimulation from the porous structure. Based on these results, we believe that the meringue-like cell-laden structure can serve as a potential biomedical scaffold, whereas the biofabrication process can be as efficient method for fabricating porous cell-laden structures for various tissue engineering applications. Geun Hyung Kim: Conceptualization (lead); data curation (equal); funding acquisition (lead); investigation (equal); project administration (lead); resources (lead); supervision (lead); writingreview and editing (lead).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.